Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that evokes a variety of cellular processes, including those that result in cell proliferation, cell morphology, tumor-cell invasion, endothelial cell chemotaxis, and angiogenesis. S1P mediates its effects on cellular behavior through the S1P receptors, a family of five cell surface G protein coupled receptors called S1P(1), S1P(2), S1P(3), S1P(4), and S1P(5), which were formerly known as EDG-1, -3, -5, -6, and -8, respectively. In addition to the S1P receptors, S1P also activates various less well-defined intracellular targets. The EDG receptors are G-protein coupled receptors (GPCRs) and on stimulation propagate second messenger signals via activation of heterotrimeric G-protein alpha (Gα) subunits and beta-gamma (Gβγ) dimers. Ultimately, this S1P-driven signaling results in cell survival, increased cell migration and, often, mitogenesis. The recent development of agonists targeting S1P receptors has provided insight regarding the role of this signaling system in physiologic homeostasis.
S1P is synthesized by the action of two enzymes, sphingosine kinase types 1 and 2 (SphK1, SphK2). These enzymes catalyze the transfer of a phosphate residue from adenosine triphosphate (ATP) to D-erythro sphingosine. SphK1 and SphK2 also catalyze the phosphorylation of reduced sphingosine (D-erythro sphinganine) and hydroxylated sphinganine (D-ribo phytosphingosine) to yield sphinganine 1-phosphate (dihydroS1P) and phytosphingosine 1-phosphate.
One example of a non-S1P agonist is the phosphorylated form of the immunomodulator, fingolimod (2-amino-2-[2-(4-octylphenyl)ethyl]propane 1,3-diol), which is an agonist of four of the five S1P receptors. Enhancing S1P tone at S1P(1) influences lymphocyte trafficking by decreasing lymphocyte egress from secondary lymphoid tissues. Consistent with the role of S1P(1) agonists in preventing lymphocyte egress from the vasculature, antagonists of some S1P(1) receptors cause leakage of the lung capillary endothelium, which suggests that S1P may be involved in maintaining the integrity of the endothelial barrier in some tissue beds.
Indeed, infection and tissue injury induce a cascade of biochemical changes that trigger reactions of the immune system, collectively referred to as an inflammatory response. The evolution of this response is based, at least in part, on enhancing vascular permeability and activation of the vascular endothelium, which allows white blood cells to efficiently circulate and migrate to the damaged site, thereby increasing their chances to bind to and destroy any antigens. The vascular endothelium is then thought to be activated or inflamed. Generally, inflammation is a welcomed immune response to a variety of unexpected stimuli, and as such it exhibits rapid onset and short duration (acute inflammation). Its persistent or uncontrolled activity (chronic inflammation) has, however, detrimental effects to the body and results in the pathogenesis of several immune diseases, such as: septic shock, rheumatoid arthritis, inflammatory bowel diseases, acute lung injury, pulmonary fibrosis, and congestive heart failure, for example: Furthermore, chronic inflammation resulting from persistent tissue injury can lead to organ fibrosis, and eventually, organ failure, as is the case in idiopathic pulmonary fibrosis, end-stage renal failure, and liver cirrhosis, for example.
During vascular injury and in inflammation thrombin is also released from the blood, and it activates thrombin receptors (PARs) expressed on endothelial surface. Thrombin and thrombin receptors regulate various endothelial functions and play a role in the response of endothelial cells to vascular injury, including inducing cytoskeletal changes resulting in cell rounding. Contraction of endothelial cells leads to increased permeability and compromises in the endothelial barrier. In contrast to the edemagenic effects of thrombin, S1P may enhance endothelial cell barrier properties.
S1P has also been shown to have a direct role in modulating several important effects on cells that mediate immune functions. Platelets, monocytes and mast cells secrete S1P upon activation, promoting inflammatory cascades at the site of tissue damage. Activation of SphK is required for the signaling responses since the ability of TNF-α to induce adhesion molecule expression via activation of Nuclear Factor Kappa B (NFκB) is mimicked by S1P and is blocked by DMS. Similarly, S1P mimics the ability of TNF-α to induce the expression of Cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2), and knock-down of SphK by RNA interference blocks these responses to TNF-α. S1P is also a mediator of calcium influx during neutrophil activation by TNF-α and other stimuli, leading to the production of superoxide and other toxic radicals. Therefore, reducing the production of S1P within immune cells and their target tissues may be an effective method to treat disorders arising from oxidative stress and abnormal inflammation. Examples of such disorders include inflammatory bowel disease, arthritis, atherosclerosis, asthma, allergy, inflammatory kidney disease, circulatory shock, ischemia-reperfusion injury, post-surgical organ failure, organ transplantation, multiple sclerosis, chronic obstructive pulmonary disease, skin inflammation, periodontal disease, psoriasis and T cell-mediated diseases of immunity.
S1P also has several effects on cells that mediate immune functions. For example, platelets, monocytes, and mast cells secrete S1P upon activation, promoting inflammatory cascades. It is believed that SphK activation is required for the related signaling responses. In addition, deregulation of apoptosis in phagocytes can be an important component of chronic inflammatory diseases. S1P has been found to protect neutrophils and macrophages in response to inflammatory stresses, such as TNF-α. Additional information regarding the role of S1P and SphK in various specific inflammatory and/or autoimmune conditions can be found in U.S. Patent Application Publication No. 2008/0167352, the disclosure of which is incorporated herein. Accordingly, inhibition of the enzymatic activity of SphK (which can reduce levels of S1P) can prevent the hyperproliferation of immune cells that are important for inflammation.
S1P also has effects on vascular contractility, vascular tone, and blood pressure control. For example, the non-S1P agonist, fingolimod produces modest hypertension in patients (2-3 mmHg in 1-yr trial). In addition, it has been found that exogenous S1P elicits a marked Ca2+- and Rho kinase-dependent pulmonary vasoconstriction in hypertensive rat lungs. Furthermore, it has been found that S1P selectively and potently constricts isolated cerebral arteries. Therefore, reducing S1P levels may be an effective method to treat disease or disorders arising from hypertension. Examples of such diseases or disorders include chronic kidney disease, pulmonary hypertension, pulmonary arterial hypertension, atherosclerosis, and stroke.
Given S1P's involvement in mediating disease pathologies associated with changes in cellular proliferation, morphology, migration, and chemotaxis, sphingosine kinases are good targets for therapeutic applications such as modulating fibrosis, tumor growth inhibition, angiogenesis, endothelial cell chemotaxis, and inflammatory and autoimmune diseases and disorders. For example, SphK1 and SphK2 have roles in affecting cell survival and proliferation. These kinases are also responsible for the equilibrium between the anti-apoptotic S1P and its pro-apoptotic metabolic precursor sphingosine and its precursor, ceramide. Thus, SphK1 and SphK2 are important drug targets.
To date, only a small number of compounds including DL-threo-dihydrosphingosine, N,N-dimethylsphingosine, and short-chain DL-erythro-sphingosine analogues, have been shown to inhibit sphingosine kinases. However, with a typical KI value of less than 10 microM, these compounds have relatively low potency. These compounds are also neither generally selective for either SphK1 or SphK2, nor are they metabolically stable in vivo. Accordingly, these compounds are not ideally suited for addressing questions concerning SphK mediated disease states.
Traditional methods of inhibiting kinases, including sphingosine kinases, have centered on targeting the ATP binding site of the kinase, a strategy that has enjoyed moderate success. However, such methods suffer from lack of selectivity across a wide array of kinases. Additionally, the amino acid sequence of the ATP binding domain of SphK1 and SphK2 is conserved across a number of diacylglycerol (DAG) kinase family members, rendering the traditional strategy problematic because it does not discriminate among kinases. By contrast, the inhibitors in the present invention are competitive with sphingosine, not with ATP, and thus are not expected to inhibit other protein and diacylglycerol kinases.
Currently, there is a need for novel, potent, and selective agents that inhibit the sphingosine substrate-binding domain of the sphingosine kinases (e.g., human SphK1 or SphK2, or both) that have enhanced potency, selectivity, and bioavailability. In addition, there is a need in the art for identification, as well as the synthesis and use, of such compounds. The present invention satisfies these needs.